U.S. patent number 4,599,231 [Application Number 06/587,983] was granted by the patent office on 1986-07-08 for synthetic hepatitis b virus vaccine including both t cell and b cell determinants.
This patent grant is currently assigned to Scripps Clinic and Research Foundation. Invention is credited to Frank V. Chisari, David R. Milich.
United States Patent |
4,599,231 |
Milich , et al. |
July 8, 1986 |
Synthetic hepatitis B virus vaccine including both T cell and B
cell determinants
Abstract
Chemically synthesized polypeptides include amino acid residue
sequences that substantially correspond to the amino acid residue
sequences of T Cell and B cell determinant portions of a natural,
pathogen-related protein, in particular, a hepatitis B virus
surface antigen (HBsAG). When administered to a host alone, as
polymers or as carrier-bound conjugates, the polypeptides induce
the proliferation of thymus-derived cells in hosts primed against
hepatitis B virus.
Inventors: |
Milich; David R. (San Diego,
CA), Chisari; Frank V. (Del Mar, CA) |
Assignee: |
Scripps Clinic and Research
Foundation (La Jolla, CA)
|
Family
ID: |
24351980 |
Appl.
No.: |
06/587,983 |
Filed: |
March 9, 1984 |
Current U.S.
Class: |
424/189.1;
530/806; 930/DIG.821; 514/21.5; 514/21.3; 514/4.3; 514/21.4;
530/324; 530/327; 930/223; 424/196.11; 424/227.1; 530/326;
530/328 |
Current CPC
Class: |
C07K
14/005 (20130101); Y10S 930/223 (20130101); Y10S
530/806 (20130101); C12N 2730/10122 (20130101); A61K
39/00 (20130101) |
Current International
Class: |
C07K
14/02 (20060101); C07K 14/005 (20060101); A61K
39/00 (20060101); A61K 039/29 (); C07K 007/06 ();
C07K 007/08 (); C07K 007/10 () |
Field of
Search: |
;260/112.5R ;424/89,88
;514/14,15,13,16 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1108051 |
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Sep 1981 |
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CA |
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0044710 |
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Jan 1982 |
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EP |
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0056249 |
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Jul 1982 |
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EP |
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0082789 |
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Jun 1983 |
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EP |
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0119342 |
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Sep 1984 |
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EP |
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84/03564 |
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Sep 1984 |
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WO |
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84/03506 |
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Sep 1984 |
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WO |
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Other References
Chem. Abstr., vol. 101, p. 108574 (1984). .
Chem. Abstr., vol. 101, p. 108640 (1984). .
Chem. Abstr., vol. 101, p. 88513 (1984). .
Chem. Abstr., vol. 99, 3895 (1983). .
The Lancet (1984) 184-187, Brown, et al. .
Nature, vol. 282, (1979) 575-582. .
Mod. Approaches Vaccines: Mol. Chem. Basis Virus, Virulence Immun.
(1983). .
Proc. Nat'l Acad. Sc., 79, 4400-4404 (1982). .
Proc. Nat'l. Acad. Sci., vol. 80, 2365-69 (1983). .
The Journal of Invest., 83, 112s-115s (1984)..
|
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Dressler, Goldsmith, Shore, Sutker
& Milnamow, Ltd.
Government Interests
The United States Government has rights in this invention pursuant
to grants awarded by the National Institutes of Health.
Claims
What is claimed is:
1. A vaccine against infection by hepatitis B virus comprising:
(a) an effective amount of at least one synthetic polypeptide
having an amino acid residue sequence taken from left to right and
in the direction from amino-terminus to carboxy-terminus selected
from the group consisting of:
SerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGlnAsn; ValCysLeuGlyGlnAsn;
CysLeuGlyGlnAsnSerGlnSerProThrSerAsnHis
SerProThrSerCysProProThrCysProGlyTyr ArgTrpMetCysLeuArgArgPheIle;
and LeuValLeuLeuAspTyrGlnGlyMetLeuProValCysProLeu;
(b) an effective amount of at least one synthetic polypeptide
having an amino acid residue sequence taken from left to right and
in the direction from amino-terminus to carboxy-terminus selected
from the group consisting of:
PheProGlySerSerThrThrSerThrGlyProCysArgThrCys
MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;
MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;
IleProGlySerThrThrThrSerThrGlyProCysLysThrCys
ThrThrProAlaGlnGlyAsnSerMetPheProSerCys;
ThrThrProAlaGlnGlyAsnSerMetPheProSerCys; and
CysProLeuIleProGlySerThrThrThrSerThrGlyPro
CysLysThrCysThrThrProAlaGlnGlyAsnSerMet PheProSerCys; and
(c) a physiologically tolerable diluent,
said vaccine when introduced into a host, being capable of inducing
the production of antibodies and the proliferation of
thymus-derived cells in the host, said antibodies immunoreacting
with said hepatitis B virus, and said vaccine protecting the host
from hepatitis B viral infection.
2. The vaccine according to claim 1 wherein said physiologically
tolerable diluent is a member of the group consisting of water,
saline and an adjuvant.
3. The vaccine according to claim 1 wherein said synthetic
polypeptides are bound to a carrier.
4. The vaccine according to claim 1 wherein said carrier is
selected from the group consisting of keyhole limpet hemocyanin,
keyhole limpet hemocyanin in incomplete Freund's adjuvant, alum,
keyhole limpet hemocyanin-alum absorbed, keyhole limpet
hemocyanin-alum absorbed-pertussis, edestin, thyroglobulin, tetanus
toxoid, tetanus toxoid in incomplete Freund's adjuvant, cholera
toxoid and cholera toxoid in incomplete Freund's adjuvant.
5. A vaccine against infection by hepatitis B virus comprising an
effective amount of a synthetic polypeptide having an amino acid
residue sequence shorter than that of hepatitis B virus surface
antigen that immunologically corresponds substantially to limited
portions of an amino acid residue sequence of the hepatitis B virus
surface antigen, said limited portions being (a) from about
positions 38 to 52 and (b) from about positions 110 to 137 from the
amino-terminus thereof, and a physiologically tolerable diluent,
said vaccine when introduced into a host, being capable of inducing
the production of antibodies and the proliferation of
thymus-derived cells in the host, said antibodies immunoreacting
with said hepatitis B virus and said vaccine protecting the host
from heptatitis B viral infection.
6. The vaccine according to claim 5 wherein the synthetic
polypeptide includes the sequences of amino acid residues taken
from left to right and in the direction from amino-terminus to
carboxy-terminus, and, represented by the formulae:
SerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGlnAsn; and
Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGly
ProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)Ala
GlnGlyThr(Asn)SerMetTyr(Phe)ProSerCys
wherein each amino acid residue in parentheses is an alternative to
the immediately preceding amino acid residue.
7. A vaccine against infection by hepatitis B virus comprising an
effective amount of a synthetic polypeptide having an amino acid
residue sequence shorter than that of hepatitis B virus surface
antigen that immunologically corresponds substantially to limited
portions of an amino acid residue sequence of the hepatitis B virus
surface antigen, said limited portions being (a) from about
positions 47 to 52 and (b) from about positions 110 to 137 from the
amino-terminus thereof, and a physiologically tolerable diluent,
said vaccine when introduced into a host, being capable of inducing
the production of antibodies and the proliferation of
thymus-derived cells in the host, said antibodies immunoreacting
with said hepatitis B virus and said vaccine protecting the host
from heptatitis B viral infection.
8. The vaccine according to claim 7 wherein the synthetic
polypeptide includes the sequences of amino acid residues taken
from left to right and in the direction from amino-terminus to
carboxy-terminus, and represented by the formulae:
ValCysLeuGlyGlnAsn; and Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGly
ProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)Ala
GlnGlyThr(Asn)SerMetTyr(Phe)ProSerCys
wherein each amino acid residue in parentheses is an alternative to
the immediately preceding amino acid residue.
9. A vaccine against infection by hepatitis B virus comprising an
effective amount of a synthetic polypeptide having an amino acid
residue sequence shorter than that of hepatitis B virus surface
antigen that immunologically corresponds substantially to a limited
portion of an amino acid residue sequence of a natural
pathogen-related protein encoded by a hepatitis B virus surface
antigen, said limited portion being from about positions 95 to 137
from the amino-terminus thereof, and a physiologically tolerable
diluent, said vaccine when introduced into a host, being capable of
inducing the production of antibodies and the proliferation of
thymus-derived cells in the host, said antibodies immunoreacting
with said hepatitis B virus and said vaccine protecting the host
from hepatitis B viral infection.
10. The vaccine according to claim 9 wherein the synthetic
polypeptide includes the sequence of amino acid residues taken from
left to right and in the direction from amino-terminus to
carboxy-terminus, and represented by the formula:
LeuValLeuLeuAspTyrGlnGlyMetLeuProValCysProLeu
Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGly
ProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)Ala
GlnGlyThr(Asn)SerMetTyr(Phe)ProSerCys
wherein each amino acid residue in parentheses is an alternative to
the immediately preceding amino acid residue.
11. A vaccine against infection by hepatitis B virus comprising an
effective amount of a synthetic multimer in a physiologically
tolerable diluent, said synthetic multimer comprising a plurality
of polypeptide repeating units including
(a) at least one amino acid residue sequence taken from left to
right and in the direction from amino-terminus to carboxy-terminus
selected from the group consisting of:
SerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGlnAsn; ValCysLeuGlyGlnAsn;
CysLeuGlyGlnAsnSerGlnSerProThrSerAsnHis
SerProThrSerCysProProThrCysProGlyTyr ArgTrpMetCysLeuArgArgPheIle;
and LeuValLeuLeuAspTyrGlnGlyMetLeuProValCysProLeu; and
(b) at least one amino acid residue sequence taken from left to
right and in the direction from amino-terminus to carboxy-terminus
selected from the group consisting of:
PheProGlySerSerThrThrSerThrGlyProCysArgThrCys
MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;
MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;
IleProGlySerThrThrThrSerThrGlyProCysLysThrCys
ThrThrProAlaGlnGlyAsnSerMetPheProSerCys;
ThrThrProAlaGlnGlyAsnSerMetPheProSerCys; and
CysProLeuIleProGlySerThrThrThrSerThrGlyPro
CysLysThrCysThrThrProAlaGlnGlyAsnSerMet PheProSerCys;
wherein at least two Cys residues are present and said synthetic
multimer contains at least one intramolecular cystine disulfide
bond formed from at least two of the Cys residues present, said
vaccine when introduced into a host, being capable of inducing the
production of antibodies and the proliferation of thymus-derived
cells in the host, said antibodies immunoreacting with said
hepatitis B virus, and said vaccine protecting the host from
hepatitis B viral infection.
12. The vaccine according to claim 11 wherein said physiologically
tolerable diluent is a member of the group consisting of water,
saline and an adjuvant.
13. The vaccine according to claim 11 wherein said synthetic
multimer is bound to a carrier.
14. The vaccine according to claim 11 wherein said carrier is
selected from the group consisting of keyhole limpet hemocyanin,
keyhole limpet hemocyanin in incomplete Freund's adjuvant, alum,
keyhole limpet hemocyanin-alum absorbed, keyhole limpet
hemocyanin-alum absorbed-pertussis, edestin, thyroglobulin, tetanus
toxoid, tetanus toxoid in incomplete Freund's adjuvant, cholera
toxoid and cholera toxoid in incomplete Freund's adjuvant.
15. The vaccine according to claim 11 wherein the intramolecular
cystine disulfide bond of said synthetic multimer is an
intrapolypeptide disulfide bond.
16. The vaccine according to claim 11 wherein the polypeptide
repeating units of said synthetic multimer are bonded together
head-to-tail through an amide bond formed between the amine group
of the amino-terminal residue of a first polypeptide repeating unit
and the carboxyl group of the carboxy-terminal residue of a second
polypeptide repeating unit.
17. The vaccine according to claim 16 wherein said synthetic
multimer contains about two to about three of said polypeptide
repeating units.
18. The vaccine according to claim 11 wherein the intramolecular
cystine disulfide bond of said synthetic multimer is an
interpolypeptide disulfide bond.
19. The vaccine according to claim 18 wherein the polypeptide
repeating units of said synthetic multimer are bonded together by
said interpolypeptide cystine disulfide bond formed between the Cys
residues of said polypeptide.
20. A vaccine against infection by hepatitis B virus comprising a
physiologically tolerable diluent having dispersed therein (i) an
effective amount of a synthetic polypeptide having an amino acid
residue sequence that immunologically corresponds substantially to
a portion of an amino acid residue sequence of the hepatitis B
virus surface antigen from about positions 110 to 137 from the
amino-terminus thereof and (ii) an effective amount of a synthetic
polypeptide having an amino acid residue sequence taken from left
to right and in the direction from amino-terminus to
carboxy-terminus, and represented by the formula:
SerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGlnAsn; said vaccine, when
introduced into a host, being capable of inducing the production of
antibodies and the proliferation of thymus-derived cells in the
host, said antibodies immunoreacting with said hepatitis B virus
and said vaccine protecting the host from hepatitis B viral
infection.
21. A vaccine against infection by hepatitis B virus comprising a
physiologically tolerable diluent having dispersed therein (i) an
effective amount of a synthetic polypeptide having an amino acid
residue sequence that immunologically corresponds substantially to
a portion of an amino acid residue sequence of the hepatitis B
virus surface antigen from about positions 110 to 137 from the
amino-terminus thereof and (ii) an effective amount of a synthetic
polypeptide having an amino acid residue sequence taken from left
to right and in the direction from amino-terminus to
carboxy-terminus, and represented by the formula:
ValCysLeuGlyGlnAsn;
said vaccine, when introduced into a host, being capable of
inducing the production of antibodies and the proliferation of
thymus-derived cells in the host, said antibodies immunoreacting
with said hepatitis B virus and said vaccine protecting the host
from hepatitis B viral infection.
22. A vaccine against infection by hepatitis B virus comprising a
physiologically tolerable diluent having dispersed therein an
effective amount of a synthetic polypeptide having an amino acid
residue sequence taken from left to right and in the direction from
amino-terminus to carboxy-terminus, and represented by the
formula:
LeuValLeuLeuAspTyrGlnGlyMetLeuProValCysProLeu
Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCysArg
(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)Ser
MetTyr(Phe)ProSerCys
wherein each amino acid residue in parentheses is an alternative to
the immediately preceding amino acid residue, said vaccine, when
introduced into a host, being capable of inducing the production of
antibodies and the proliferation of thymus-derived cells in the
host, said antibodies immunoreacting with said hepatitis B virus
and said vaccine protecting the host from hepatitis B viral
infection.
Description
DESCRIPTION
TECHNICAL FIELD
The present invention relates to chemically synthesized
polypeptides having amino acid residue sequences that substantially
correspond to the amino acid residue sequences of T cell and B cell
determinant portions of a natural, pathogen-related protein, in
particular, a hepatitis B virus surface antigen (HBsAg). When
administered to a host alone, as polymers or as carrier-bound
conjugates, the polypeptides induce the proliferation of
thymus-derived cells in hosts primed against hepatitis B virus.
BACKGROUND
The present invention relates to the production of novel synthetic
antigens based upon information derived from DNA and/or protein
sequences and to the use of those antigens in the production of
vaccines, diagnostic reagents, and the like. More specifically,
this invention relates to synthetic antigenic polypeptides, which
when used alone, as a polymer or upon coupling to a carrier,
immunologically correspond to a T cell and B cell determinant
portion of hepatitis B virus surface antigen (HBsAg).
Viral hepatitis continues to rank as one of the most important
unconquered diseases of mankind. The general term, viral hepatitis,
refers principally to hepatitis A (infectious hepatitis) and to
hepatitis B (serum hepatitis), although other known viruses such as
yellow fever virus, Epstein-Barr virus and cytomegalovirus can
cause hepatitis in man. Hepatitis is particularly known for its
focal attack on the liver (Greek, hepar), but the disease also
influences other organs.
In 1965, Blumberg discovered an antigen circulating in the blood of
certain human beings [J. Am. Med. Assoc., 191, 541 (1965) and Ann.
Int. Med., 66, 924 (1967)]. This substance was subsequently found
by Prince to be the surface antigen of hepatitis B virus (HBsAg)
that is produced in abundance by individuals who are chronically
infected with the agent [Proc. Natl. Acad. Sci. (USA), 60, 814
(1968)].
HBsAg has been the subject of extensive immunochemical
characterization. Serologic studies show that several strains of
the hepatitis B virus (HBV) have one or more determinants in
common, which is designated a. Each strain also has two other
determinants: either d or y and either w or r. Thus, there are four
possible types of the virus: adw, ayw, adr and ayr. The specificity
of HBsAg is associated with a single polypeptide [Gold et al., J.
Immunol., 117, 1404 (1976) and Shih et al., J. Immunol., 120, 520
(1978)], the entire 226 amino acid sequence of which is established
from the nucleotide sequence of the S gene [Tiollais et al.,
Science, 213, 406 (1981)] of HBV [Valenzuela et al., Nature
(London), 280, 815 (1979); Galibert et al., Nature (London), 281,
646 (1979) and Pasek et al., Nature (London), 282, 575 (1979)].
There is an urgent need for a hepatitis B vaccine for groups which
are at an increased risk of acquiring this infection. These groups
include health care and laboratory personnel, and individuals
requiring (1) maintenance hemodialysis; (2) repeated blood
transfusions or the administration of blood products; (3) treatment
with immunosuppressive or cytotoxic drugs and (4) treatment for
malignant diseases and disorders associated with depression of the
immune response. In addition, a vaccine is needed for individuals
living in certain tropical areas where hepatitis B infection is
prevalent.
Hepatitis A and B viruses, however, do not multiply significantly
in cell culture, and there is no current source of laboratory
propagated virus for vaccine preparation. Indeed, there has been a
repeated failure to transmit hepatitis B virus (HBV) serially in
tissue or organ cultures which has hampered progress towards the
development of a conventional vaccine [Zuckerman, Amer. J. Med.
Sci., 270, 205 (1975)].
Classically, a vaccine is manufactured by introducing a killed or
attenuated organism into the host along with suitable adjuvants to
initiate the normal immune response to the organism while,
desirably, avoiding the pathogenic effects of the organism in the
host. That approach suffers from several well known limitations.
These vaccines are complex and include not only the antigenic
determinant of interest but many related and unrelated deleterious
materials, any number of which may, in some or all individuals,
induce an undesirable reaction in the host.
For example, vaccines produced in the classical way may include
competing antigens which are detrimental to the desired immune
response, antigens which include unrelated immune responses,
nucleic acids from the organism or culture, endotoxins and
constituents of unknown composition and source. These vaccines,
generated from complex materials, inherently have a relatively high
probability of inducing competing responses even from the antigen
of interest.
In the past, antigens have been obtained by several methods
including derivation from natural materials, coupling of a hapten
to a carrier and by recombinant DNA technology. Sela et al. [Proc.
Nat. Acad. Sci. (USA), 68, 1450 (1971); Science, 166, 1365 (1969);
and Adv. Immun., 5, 129 (1966)] have also described certain
synthetic antigens.
Certain "synthetic" antigens have been prepared by coupling small
molecules (for example, dinitrophenol) to carriers (such as bovine
serum albumin), thus producing antigens which cause the production
of antibody to the coupled small molecule. The carrier molecule is
often necessary because the small molecule itself may not be
"recognized" by the immune system of the animal into which it is
injected. This technique has also been employed in isolated
instances to prepare antigens by coupling polypeptide fragments of
known proteins to carriers, as described in the above-referenced
Sela et al articles.
While this hapten-carrier technique has served the research
community well in its investigations of the nature of the immune
response, it has not been of significant use in the production of
antigens which would play a role in diagnostic or therapeutic
modalities. One reason for that deficiency is that to select and
construct a useful antigenic determinant from a pathogen (e.g.,
hepatitis B virus) by this technique, one must determine the entire
protein sequence of the pathogen to have a reasonable chance of
success. Because of the difficulty of this task, it has rarely, if
ever, been done.
Recombinant DNA technology has opened new approaches to vaccine
technology and has the advantage that the manufacture begins with a
monospecific gene; however, much of this advantage is lost in
actual production of antigen in E. coli, or other organisms. In
this procedure, the gene material is introduced into a plasmid
which is then introduced into E. coli which produces the desired
protein, along with other products of the metabolism, all in
mixture with the nutrient. This approach is complicated by the
uncertainty as to whether the desired protein will be expressed in
the transformed E. coli.
Moreover, even though the desired protein may be produced, there is
uncertainty as to whether or not the protein can be harvested or
whether it will be destroyed in the process of E. coli growth. It
is well known, for example, that foreign or altered proteins are
digested by E. coli. Even if the protein is present in sufficient
quantities to be of interest, it must still be separated from all
of the other products of the E. coli metabolism, including such
deleterious substances as undesired proteins, endotoxins, nucleic
acids, genes and unknown or unpredictable substances.
Finally, even if it were possible (or becomes possible through
advanced, though necessarily very expensive, techniques) to
separate the desired protein from all other products of E. coli
metabolism, the vaccine still comprises an entire protein which may
include undesirable antigenic determinants, some of which are known
to initiate adverse responses. Indeed, it is known that certain
proteins which could otherwise be considered as vaccines include an
antigenic determinant which induces serious cross reference or side
reactions that prevent the use of the material as a vaccine.
It is also possible, using hybridoma technology, to produce
antibodies to viral gene products. Basically, hybridoma technology
allows one to begin with a complex mixture of antigens and to
produce monospecific antibodies later in the process. In contrast,
the present invention is the reverse process, in that we start with
a relatively high purity antigenic determinant and thus avoid the
necessity for purification of the desired antigenic product.
Hybridoma antibodies are known to exhibit low avidity and low
binding constants, and therefore, have limited value. Moreover, in
hybridoma technology, one must rely on the production of the
antibody by cells which are malignant, with all of the attendant
concerns regarding separation techniques, purity and safety.
Hybridoma production relies upon tissue culture or introduction
into mice, with the obvious result that production is costly and
there is an inherent variability from lot to lot.
In addition, it is difficult to make hybridomas that secrete
antibodies to molecules which comprise only a small percentage of
the complex mixture with which one must start, or which are poorly
immunogenic and are overshadowed by stronger, dominant
antigens.
Previous studies by Arnon et al., Proc. Nat. Acad. Sci. (USA), 68,
1450 (1971), Atassi, Immunochemistry, 12, 423 (1975) and Vyas et
al., Science, 178, 1300 (1972) have been interpreted by those
authors to indicate that short linear amino acid sequences are, in
general, unlikely to elicit antibodies reactive with the native
protein structure. It was thought that for most regions of most
molecules, antigenic determinants resulted from amino acid residues
well separated in the linear sequence but close together in the
folded protein. The exact three dimensional conformation of the
polypeptides used to elicit antibodies was thought to be critical
in most cases, even for those antigens involving amino acids close
together in a sequence.
For example, Sela thought it necessary to synthesize a rather
elaborate loop structure to elicit an anti-lysozyme response.
Atassi engineered many elaborate molecules, each intended to mimic
the tertiary structure of the target protein. And Vyas concluded
that the three dimensional conformation of hepatitis B surface
antigen was a critical factor in eliciting antibodies reactive with
that native structure.
Sutcliffe et al., Nature, 287, 801 (1980) discovered that
antibodies to linear polypeptides react with native molecules, and
recent investigations have shown that relatively short chemically
synthesized polypeptides can elicit antibodies reactive with almost
any region of an exposed surface of a protein [Green et al., Cell,
28, 477 (1982)]. Moreover, since amino-acid sequences can now be
determined rapidly with nucleic acid sequencing technology,
synthetic polypeptides can be synthesized to make vaccines of a
precision not previously possible. Thus, elaborate biosyntheses are
unnecessary, uneconomical and obsolete.
U.S. Pat. No. 4,415,491 to Vyas discloses a series of peptides that
correspond to the a determinant of hepatitis B virus surface
antigen. Although no data is presented concerning the protection of
a host, the peptides are described as being useful in a hepatitis
vaccine preparation.
Current vaccines for HBV consist of subviral components of the
virus surface coat (HBsAg) purified from the plasma of chronically
HBV-infected donors and inactivated [McAuliffe et al., Rev. Infect.
Dis., 2, 470 (1980)]. Clinical trials have demonstrated the safety
and efficacy of current HBsAg vaccines but such vaccines are
limited in supply and are relatively expensive, particularly for
those countries with the highest incidence of HBV disease.
Chemically synthesized polypeptides, therefore, offer considerable
advantages in terms of cost and safety of HBV vaccination
programs.
It is known that antisera to synthetic polypeptides predicted from
the nucleotide sequence of various regions of the S gene of HBV
react with native HBsAg by radioimmunoprecipitation [Lerner et al.,
Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981)] and commercial
solid-phase radioimmunoassays for anti-HBsAg [Gerin et al., in
Viral Hepatitis, Szmuness et al (eds.), 49-55 (1982)].
It has been recently determined that a pathogen-related protein can
be immunologically mimicked by the production of a synthetic
polypeptide whose sequence corresponds to that of a determinant
domain of the pathogen-related protein. Such findings are reported
by Sutcliffe et al., Nature, 287, 801 (1980) and Lerner et al.,
Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981).
Moreover, Gerin et al., Proc. Natl. Acad. Sci. (USA), 80, 2365
(1983) have recently shown limited protection from hepatitis B
virus upon immunization with carrier bound-synthetic polypeptides
having amino acid sequences that correspond to the amino acid
sequence of a determinant portion of HBsAg; in particular, residues
110-137.
The construction of a synthetic HBsAg vaccine, however, may require
in addition to synthetic polypeptides corresponding to B cell
(antibody-producing) epitopes, synthetic polypeptides corresponding
to non-overlapping T cell determinants.
By way of further background, three cellular components of the
immune system are B cells (bursaor bone marrow-derived
lymphocytes), T cells (thymus-derived lymphocytes) and macrophages.
B cells circulate in the blood and the lymph fluid and are involved
in the production of antibodies. T cells amplify or suppress the
response by B cells.
Macrophages, on the other hand, are involved in presenting and
concentrating antigens to B and T cells. Moreover, macrophages
secrete several biologically active mediators that regulate the
type and magnitude of both T and B cell responses either by
enhancing or suppressing cell division or differentiation.
Macrophages are nonspecific and react against any foreign antigen.
T and B cell, however, are antigen-specific and react via cell
membrane receptors that are specific for the particular
antigen.
In mice, the in vivo antibody production to HBsAg is regulated by
at least 2 immune response (Ir) genes, one in the I-A subregion
(Ir-HBs-1) and one in the I-C subregion (Ir-HBs-2) of the murine
H-2 complex. It is observed that immunization with a chemically
synthesized peptide corresponding to the d determinant did not
distinguish between high and non-responder murine strains. Milich
et al., J. Immunol., 130, 1401 (1983). This suggests that
Ir-restriction may occur through T cell recognition of additional,
perhaps nonoverlapping, regions of the molecule.
The linkage between major histocompatibility complex and the
regulation of immune responsiveness to HBsAg in mice has been
extended to the human immune response by the report of an
association between HLA-DR phenotype and nonresponsiveness to a
recent trial HBsAg vaccine. Thus, the construction of synthetic
HBsAg vaccine may require, in addition to B cell epitopes, a
sufficient diversity of T cell determinants to accommodate the
genetic variation in epitope recognition of an outbred human
population.
The following information would be very valuable in developing a
synthetic HBsAg vaccine: (1) whether synthetic peptide fragments
representing a highly restricted region of the native HBsAg (i.e.,
about 6 amino acids) can induce a T cell proliferative response,
which, as with native HBsAg, is regulated by H-2 linked genes; (2)
whether T cell recognition sites overlap with antibody binding
sites; (3) whether multiple T cell recognition sites exist on HBsAg
and if so whether the site(s) recognized depend on the H-2 genotype
of the responding strain; (4) whether the T cell site(s) recognized
determine the specificity and quality of the humoral response; and
(5) whether human HBsAg-primed T cells are activated by the same
determinants that induce T cell proliferation in mice.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to certain synthetic polypeptides
that have special characteristics and properties, and to products
and methods utilizing those synthetic polypeptides.
Throughout this application, the terms "peptide" and "polypeptide"
are used interchangeably. As used herein, the term "synthetic
polypeptide" means a chemically built-up, as compared to a
biologically built and degraded, chain of amino acid residues that
is free of naturally occurring proteins and fragments thereof. Such
synthetic polypeptides can induce the production of
anti-polypeptide antibodies in a host.
A synthetic polypeptide in accordance with this invention has an
amino acid residue sequence that is shorter than that of hepatitis
B virus surface antigen but includes an amino acid residue sequence
that corresponds immunologically to that of at least one
determinant portion of hepatitis B virus surface antigen
(HBsAg).
The polypeptide, when used alone, as a polymer (synthetic multimer)
or bound to a carrier such as keyhole limpet hemocyanin (KLH) or
the like as a conjugate and introduced in an effective amount as a
vaccine in a physiologically tolerable diluent such as water,
saline and/or an adjuvant into a host animal, can induce the
production of antibodies and the proliferation of thymus-derived
cells in the host.
The vaccine is prepared by providing one or more of the following
polypeptides, a polymer thereof or a carrier-bound conjugate
thereof and dissolving or dispersing an effective amount of the
polypeptide in a physiologically tolerable diluent.
Preferred sequences of synthetic polypeptides, for use in a
vaccine, comprise amino acid residue sequences (or a portion
thereof) of B cell determinant portions of HBsAg (also referred to
herein as B cell-stimulating and priming portions) taken from left
to right and in the direction from the amino-terminus to the
carboxy-terminus including: ##STR1## wherein each amino acid
residue in parentheses is an alternative to the immediately
preceding amino acid residue, and the numerals in parentheses above
particular amino acid residues in the above sequences identify
positions of the particular amino acid residue relative to the
amino-terminus of the hepatitis B virus surface protein. Such
polypeptides induce the production of antibodies that can
immunoreact with hepatitis B virus and protect a host from
infection.
Preferred sequences of synthetic polypeptides, for use in a
vaccine, also include amino acid residue sequences (or portions
thereof) of T cell determinant portions of HBsAg that induce T cell
to proliferate (also referred to herein as T cell-proliferating
portions) taken from left to right and in the direction from the
amino-terminus to the carboxy-terminus including: ##STR2## wherein
the numbers in parentheses above particular amino acid residues in
the above sequences identify positions of the particular amino acid
residue relative to the amino-terminus of the hepatitis B virus
surface protein.
Each of the above synthetic polypeptides can be used in a monomeric
form alone or conjugated to a carrier molecule such as KLH or
tetanus toxoid. The synthetic polypeptides can also be used in a
multimeric form.
When utilized in multimermic form, each polypeptide is one of a
plurality of repeating units of a multimer. In one embodiment, the
multimer contains at least two of the polypeptides bonded together
head-to-tail through an amide bond formed between the amine group
of the amino-terminus of one polypeptide and the carboxyl group of
the carboxy-terminus of the second polypeptide. In another
multimeric embodiment, the polypeptide is one of a plurality of
repeating units of a polymer whose polypeptide repeating units are
bonded together by interpolypeptide cystine disulfide bonds formed
between the Cys residues of the polypeptide repeating units.
In another embodiment, the present invention includes a diagnostic
system for determining the presence of cell-mediated immune
responsiveness to HBsAg and the presence of a hepatitis B virus
antigen in a host comprising a synthetic polypeptide as described
above that has an amino acid residue sequence that corresponds to
the amino acid sequence of a T cell determinant of HBsAg. The
polypeptide, when administered to a host intradermally in an
effective amount and in physiologically tolerable diluent, is
capable of inducing the proliferation of thymus-derived cells in
the host. The proliferation is indicated by erythema (redness) and
induration (hardening of the skin) at the site of intradermal
administration.
Methods are also disclosed for inducing the proliferation of
thymus-derived cells in a host previously immunized to hepatitis B
virus and for determining the presence of a hepatitis B virus
antigen in a host. The methods include the steps of providing a
T-cell proliferating polypeptide as discussed herein and
administering intradermally an effective amount of the polypeptide
to the host in a physiologically tolerable diluent according to the
latter method, the proliferation of thymus-derived cells and the
presence of a hepatitis B virus antigen in the host is indicated by
erythema and induration at the site of intradermal
administration.
The present invention provides several advantages and benefits. One
advantage of the present invention is that use of a synthetic
polypeptide obviates the need for the presence of its corresponding
intact protein. The polypeptide itself provides a vaccine
sufficient to protect the host from disease. Consequently,
impurities such as cellular debris and toxins that are associated
with the production of usable amounts of viral proteins from
bacteria are absent from the product of this invention.
Moreover, a synthetic hepatitis B virus vaccine having both B cell
and T cell determinants obviates the need to select a carrier
appropriate for use in humans to stimulate the proliferation of
thymus-derived cells in the recipient.
Another benefit of the present invention is that antibodies in
antisera raised to the synthetic polypeptide immunoreact with and
can be used to detect the presence of antigenic proteins and
polypeptides associated with hepatitis B virus.
Still further advantages and benefits of the present invention will
become apparent to those skilled in the art from the detailed
description, Examples and claims which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings, which constitute a portion of this disclosure:
FIG. 1 illustrates the 226 amino acid sequence of the HBsAg/ayw
protein as translated by Pasek et al., Nature, 282, 575 (1979) from
the nucleic acid sequence. Regions of the protein selected for
synthesis according to the present invention are indicated by bold
underlining. Residues that are not the same in the three published
nucleotide sequence determinations are lightly underlined [Pasek et
al., Id.; Valenzuela et al., Nature, 280, 815-819 (1979); and
Galibert et al., Nature, 281, 646-650 (1979)]. The following single
letter and three letter codes (See FIG. 2) correspond to the
indicated amino acids--A, Ala (L-Alanine); C, Cys (L-Cysteine); D,
Asp (L-Aspartic acid); E, Glu (L-Glutamic acid); F, Phe
(L-Phenylalanine); G, Gly (Glycine); H, His (L-Histidine); I, Ile
(L-Isoleucine); K, Lys (L-Lysine); L, Leu (L-Leucine); M, Met
(L-Methionine); N, Asn (L-Asparagine); P, Pro (L-Proline); Q, Gln
(L-Glutamine); R, Arg (L-Arginine); S, Ser (L-Serine); T, Thr
(L-Threonine); V, Val (L-Valine); W, Trp (L-Tryptophan); and Y, Tyr
(L-Tyrosine).
FIG. 2 illustrates the amino acid sequences of polypeptides
designated 1, 5, 5a, 6, 49, 49a, 72, 72a and 73 using the
conventional three letter code for each amino acid. These sequences
are read from left to right and in the direction from the
amino-terminus to the carboxy-terminus of the polypeptide.
Polypeptides 1, 5, 5a and 6 correspond to residues 48-81, 38-52,
47-52 and 95-109, respectively, of HBsAg. Polypeptides 49 and 72
correspond to residues 110-137 of HBsAg (peptide 73 corresponds to
residues 107-137) as predicted from the S gene nucleotide sequence
of HBV DNA from an ayw donor (polypeptide 49) [Galibert et al.,
Nature (London), 281, 646-650 (1979)] and an adw donor (polypeptide
72 and 73) [Valenzuela et al., Nature (London), 280, 815-819
(1979)]. The underlined residues in polypeptides 72 and 73 indicate
positions of amino acid variability between those sequences and
that of polypeptide 49. Polypeptides 49a and 72a consist of the
C-terminal 12 amino acids of polypeptides 49 and 72, respectively
(residues 125-137).
FIG. 3 illustrates the mouse C.sub.3 H.Q strain T cell
proliferative responses in popliteal lymph node cells primed by
HBsAg (ad or ay subtype) induced in vitro by: native HBsAg; P25
(the 1-226 residue subunit of HBsAg); the following tryptic
fragments of P25: P25-1 (residues 1-122) and P25-2 (residues
123-226), and P73, P72, P49, P6, P5, P5a and P2 (residues 140-148).
As used herein, the letter "P" before a number means "peptide" or
"polypeptide". Proliferation was determined by incorporation of
tritiated thymidine (.sup.3 HTdR) into cellular DNA, and was
expressed as a percent response elicited by the immunogen. The
immunogen HBsAg/ad elicited a proliferation which produced 20,477
counts per minute (cpm) and the immunogen HBsAg/ay elicited a
proliferation which produced 33,000 cpm.
FIG. 4 illustrates the mouse B10.A strain T cell proliferative
responses induced by: native HBsAg, P25 (the 1-226 residue); the
following tryptic fragments of P25: P25-1 (residues 1-122) and
P25-2 (residues 123-226); and P73, P72, P49, P6, P5, P5a and P2.
Proliferative response doses and means of measurement were the same
as in FIG. 3. The proliferative responses elicited by immunogens
HBsAg/ad and HBsAg/ay were 8724 cpm and 11,444 cpm, respectively.
Details of the assays of FIGS. 3 and 4 are provided in Sections IV
and V.
DETAILED DESCTIPTION OF THE INVENTION
I. Introduction
Synthetic polypeptides having amino acid residue sequences that
substantially correspond to the amino acid sequences of the d (P72)
and y (P49) determinants of HBsAg have been synthesized by Lerner
et al., Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981). These
polypeptides possess the antigenic specificity of the native
determinants as demonstrated by their ability to bind anti-native
HBsAg antibodies. In addition, it has been demonstrated that
immunization with P49 conjugated to keyhole limpet hemocyanin (KLH)
induces a high-titered anti-y response in a murine inbred responder
strain. Milich et al., J. Immunol., 130, 1401 (1983).
However, immunization with free (unconjugated) P49 induces little
or no anti-y production. Similarly, free P72 induces a very minimal
anti-d response. Indeed, the reduced immunogenicity of unconjugated
(relative to conjugated) synthetic peptide analogues of HBsAg has
been encountered by numerous investigators.
Therefore, protein carrier molecules such as KLH and tetanus toxoid
have been used as a means of providing nonspecific T cell helper
function for these synthetic determinants.
In order to construct a synthetic HBsAg vaccine possessing both T
cell and B cell determinants, it is first necessary to identify the
T cell and B cell determinants of HBsAg.
It is known that the murine immune response to HBsAg is regulated
by H-2-linked Ir genes, and that this regulation is expressed at
the T cell level. Nonresponder haplotypes are characterized by a
defect in T-helper cell function, whereas HBsAg-specific B cell
repertoirs are intact. In addition to the reduced immunogenicity of
free, unconjugated synthetic peptide analogues of HBsAg,
immunization with P72 (residues 110-137) or P49 (residues 110-137)
did not distinguish between high responder and nonresponder
strains.
These results indicate that P72 and P49 represent B cell epitopes
of the native structure, but lack the appropriate T cell
determinants.
Thus, immunization with these B cell epitopes alone does not
generate the necessary Ir-restricted, T cell helper function.
A number of HBsAg synthetic peptides were screened as described
herein in an HBsAg-specific T cell proliferative assay in order to
identify T cell determinants. Mice were immunized in vivo with
native HBsAg/ad or HBsAg/ay, and popliteal lymph node (PLN) cells
were harvested and challenged in vitro with either native HBsAg or
a series of synthetic polypeptides.
At least three polypeptides were identified that stimulate
HBsAg-primed PLN cells to proliferate in vitro. In particular,
polypeptides P5 (residues 38-52), P5a (residues 47-52) and P6
(residues 95-109) of FIGS. 1 and 2 stimulate T cell proliferation
of murine PLN cells primed in vivo with HBsAg of the ad or ay
subtype.
Moreover, at least polypeptide P1 (residues 48-81) and polypeptide
P5 induce T cell proliferation in human peripheral blood
lymphocytes (PBL).
It should be noted that P1, P5, P5a and P6 do not induce the
production of antibodies cross-reactive with native HBsAg nor do
they bind native anti-HBs antibodies. Conversely, P72 and P49 do
not induce (or at best induce only minimal) T cell proliferation,
yet bind anti-HBs of the appropriate specificity, and provide some
protection against hepatitis B disease.
These results indicate the existence of distinct loci for T cell
and B cell determinants on the same HBsAg polypeptide. Use of a
synthetic T cell determinant with a B cell determinant, preferably
a synthetic B cell determinant, according to the present invention
provides a potent synthetic antigen.
Previous genetic analysis of the immune responses to HBsAg in H-2
congenic, recombinant murine strains predicts the existence of a
"carrier-determinant" on HBsAg, since a dominant influence on the
immune response to all HBsAg determinants maps to a single Ir gene
locus. Polypeptides 5, 5a and 6 correspond to such a
carrier-determinant on the native molecule. These polypeptides
function as intrinsic carriers and provide functional T cell help
for any and all synthetic B cell epitopes to which they are
coupled.
Thus, one aspect of the present invention is directed to vaccines
that contain as an active ingredient an effective amount of a T
cell-proliferating polypeptide described herein, e.g. at least one
of polypeptides 1, 5, 5a and 6. Such a vaccine may be introduced
into a host animal (or a human) after that animal has been
immunized with (primed to) a HBsAg B cell activator such as the
complete HBsAg molecule or polypeptides such as those denominated
49, 49a, 72 and 72a. More preferably, the T cell-proliferating
polypeptide of this invention is administered to the host animal
along with a priming, B cell-stimulating immunogen such as
polypeptides 49, 49a, 72 and 72a.
The more preferred T cell-proliferating and B cell-stimulating and
priming polypeptides may be introduced into the host as separate
entities of one vaccine wherein each is linked to its own carrier
or as a homopolymer of active polypeptide repeating units. More
preferably, both types of polypeptide are linked to a single
carrier and thereby constitute a single active entity in the
vaccine. A synthetic HBsAg vaccine containing co-polymerized
polypeptide repeating units with amino acid sequences that
substantially correspond to amino acid sequences of T cell and also
B cell determinants of the native molecule is clearly a still more
preferred approach rather than attempting to select appropriate
protein carrier molecule for immunization into human subjects.
Moreover, enhancement of the immunogenicity of synthetic
polypeptides related to HBsAg is a fundamental aspect in the
development of a synthetic HBsAg vaccine. The highly immunogenic
synthetic HBsAg vaccine described herein has desirable medical as
well as economic advantages as compared to the current human
plasma-derived vaccines.
II. Discussion
The data from this study demonstrate that limited regions of the
hepatitis B surface antigen (HBsAg) molecule; in particular,
residues 48 to 81 (which correspond to synthetic peptide P1),
residues 38 to 52 (which correspond to synthetic peptide P5),
residues 95 to 109 (which correspond to synthetic peptide P6) and
residues 47 to 52 (which correspond to synthetic peptide P5a) are
sites that are preferentially recognized by HBsAg-primed T
cells.
Although synthetic peptides P1, P5, P5a and P6 induce T cell
proliferative responses, these peptides do not correspondingly
induce or bind antibodies that recognize the native molecule. This
illustrates the disparity in determinant specificity that can exist
between B and T cells in response to complex protein antigens.
Such disparity has been observed in a variety of antigenic systems
as described in the following references: Senyk et al., J. Exp.
Med., 133, 1294 (1971); Thomas et al., J. Immunol., 126, 1095
(1981); Berkower et al., Proc. Natl. Acad. Sci. (USA), 79, 4723
(1982); Kipps et al., J. Immunol., 124, 1344 (1980). In contrast,
other investigators have demonstrated similar T and B cell receptor
specificities for antigens as described in the following
references: Twining et al., Mol. Immunol., 18, 447 (1981); Rajewsky
et al., Eur. J. Immunol., 4, 111 (1974); Becker et al., Eur. J.
Immunol., 5, 262 (1975). Any assumption, however, that T and B cell
recognition sites never or always overlap is therefore overly
simplistic.
With reference to HBsAg, C.sub.3 H.Q (H-2.sup.q), or simply
"C.sub.3 H.Q", and B10.T(6R)(H-2.sup.q) or "B10.T(6R)", murine
strains preferentially recognize the amino-terminal fragment of
HBsAg [in particular, residues 1-122 of the P25 HBsAg polypeptide
subunit (P25-1)] and the constituent peptides P5, P5a and P6.
Murine strains C.sub.3 H.Q and B10.T(6R) are referred to herein as
"responder strains" or a "high responder strains" based on the
degree of the proliferative response.
The proliferative response of B10.A (H-2.sup.a), or "B10.A", murine
strain T cells, on the other hand, is directed almost exclusively
to the carboxy-terminal fragment of HBsAg [specifically, residues
123-226 of P25 (P25-2)] and to the P72 synthetic peptide, which
also serve as antibody binding sites on HBsAg. Murine strain B10.A
is referred to as an "intermediate responder strain" because the
proliferative response is less than that of C.sub.3 H.Q or
B10.T(6R).
Therefore, multiple T cell recognition sites appear to exist on
HBsAg and the selective activation of T proliferating cells is
dependent on the murine major histocompatibility complex (H-2)
haplotype of the responding strain. A similar preferential
selection of T cell epitopes in a hapten-carrier system controlled
by I-region genes in the murine H-2 complex has been reported in
Seman et al., J. Immunol., 129, 2082 (1982).
The humoral anti-HBsAg response is regulated by at least two immune
response (Ir) genes. One of the genes is in the I-A subregion
(Ir-Hbs-1) and the other is in the I-C subregion (Ir-HBs-2) of the
murine H-2 complex. The Ir-Hbs-1 regulates the response to all
HBsAg determinants; whereas, the influence of the Ir-HBs-2 is
subtype-specific. (For a general description of Ir genes and
subregions see Bach, Genetic Control of Immune Responses in
Immunology, ch. 24, pages 677-703 (John Wiley & Sons, New York
1982) which description is incorporated herein by reference).
In the strains used herein, a positive T cell proliferative
response to the amino-terminal fragment of the HBsAg P25
polypeptide subunit P25-1 and to synthetic peptides P5a or P6
indicated an enhanced anti-HBs antibody production to all HBsAg
determinants. In contrast, the T cell proliferative pattern of the
B10.A murine strain corresponds to reduced primary anti-HBs
antibody production which is limited to subtype specificity.
A site or sites on the amino-terminal fragment of synthetic
peptides P5, P5a and P6 serves as a T cell "carrier-determinant"
recognized by T helper cells capable of providing functional help
to B cell clones specific for the a, d and y epitopes and
restricted by the I-A subregion. In the absence of recognition of
the "carrier-determinant," the influence of subtype-specific helper
or suppressor T cells restricted by the I-C subregion is observed.
Since the B10.A strain produces a minimal secondary anti-a antibody
response, subtype-specific T cells may also provide help to B cell
clones specific for the conformational a-epitope.
These observations have important implications in terms of the
development of a synthetic HBsAg vaccine; especially in view of the
possibility that human HBsAg-primed T cells may recognize the same
epitopes as murine T cells.
In particular, the linkage between the major histocompatability
complex and the regulation of immune responsiveness to HBsAg in
mice has been extended to the human immune respone. Walker et al.,
Proc. Amer. Assoc. Blood Banks, 4 (1981) have reported an
association between a particular phenotype at the DR gene locus of
the human major histocompatibility complex (HLA-DR) and
nonresponsiveness to a recent trial HBsAg vaccine.
Thus, the construction of a synthetic HBsAg vaccine preferably
includes, in addition to B cell determinants, a sufficient
diversity of T cell determinants to accommodate the genetic
variation in epitope recognition of an outbred human
population.
IV. Results
A. Identification of Murine B Cell Epitopes
The polypeptide sequences of hepatitis B surface antigen (HBsAg)
that induce the production of and bind to murine anti-HBsAg
antibodies were identified.
Twelve polypeptide sequences of HBsAg group a subtype yw
(HBsAg/ayw) were selected for synthetic polypeptide synthesis.
These polypeptides are denominated P1, P2, P3, P4, P5, P5a, P6,
P49, P49a, P72, P72a and P73 and are illustrated in FIG. 1.
The peptides were chemically synthesized by solid-phase methods as
described herein in Section VI and as described in greater detail
in Merrifield et al., J. Am. Chem. Soc., 85, 2149 (1963) and
Houghten et al., Int. J. Peptide Protein Research, 16, 311 (1980).
Anti-polypeptide antibodies specific for each of the synthetic
peptides were produced when the synthetic polypeptides were coupled
to KLH and introduced into rabbits as a vaccine that also included
water and an adjuvant.
Pooled purified preparations of HBsAg group a subtype d (HBsAg/ad)
and HBsAg group a subtype y (HBsAg/ay) were obtained from Dr.
Robert Louie (Cutter Laboratories, Berkeley, California). The
antibodies to the synthetic peptides were analyzed for reactivity
to HBsAg/ad and HBsAg/ay by a hemagglutination assay (HA) as
described herein. The ability of the solid-phase polypeptides to
bind murine anti-native HBsAg antibodies of d or y specificity was
also determined as described below.
Polypeptides P73 (residues 107-137), P72 (residues 110-137) and
P72a (residues 125-137) induced the production of antibodies that
were cross-reactive with native HBsAg of the ad subtype.
Polypeptides P49 (residues 110-137) and P49a (125-137), on the
other hand, induced the production of antibodies that were
cross-reactive with native HBsAg primarily of the ay subtype. (See
Table 1).
TABLE 1 ______________________________________ Identification of B
Cell Epitopes On Synthetic Peptide Analogues of HBsAg Anti-peptide
reactivity Anti-Native HBs Reactivity With Native HBsAg With
Solid-Phase Peptides.sup.2 HA Titer RIA Titer Peptide.sup.1 HBsAg/
--ad HBsAg/ --ay Anti-HBs/ -d.sup.3 Anti-HBS/ -y
______________________________________ P73 1:1280 1:40 1:512 1:32
P72 1:160 0 1:1024 0 P72a 1:160 0 ND.sup.4 ND P49 1:80.sup.5 1:160
1:32 1:128 P49a 0 1:160 0 1:64 P6 0 0 0 0 P5 0 0 1:8 0 P5a 0 0 0 0
P4 0 0 1:4 0 P3 0 0 1:16 1:8 P2 0 0 0 0 P1 0 0 1:16 0
______________________________________ .sup.1 Antipeptide antisera
were produced in rabbits; and all peptides were conjugated to
keyhole limpet hemocyanin (KLH) with the exception of P73, P72 and
P1. Antipeptide antisera prepared in mice [Milich et al., J.
Immunol., 130, 1401 (1983)] and chimpanzees [Gerin et al., Proc.
Natl. Acad. Sci. (USA), 80, 2365 (1983)] demonstrate the same
specificities fo native HBsAg. .sup.2 Peptides (5 micrograms per
well) were adsorbed to polystyrene microtiter plates. .sup.3
AntiHBs/-d and -y were produced by immunizing B10.S (9R) mice with
HBsAg/--ad or HBsAg/--ay, respectively. This H2 recombinant strain
produces only a subtypespecific antibody response. .sup.4 ND = Not
determined. .sup.5 Not specific for the common -a determinant.
Polypeptides P72 and P72a correspond in amino acid residue
positions to polypeptides P49 and P49a, but contain the amino acid
substitutions shown in FIGS. 1 and 2. Although anti-P49 antibodies
reacted with both subtypes of HBsAg, HA inhibition analysis
demonstrated the cross-reactivity was not directed to the common
"a"-determinant, but rather to a determinant present on the native
HBsAg/ad, P49 and P72 but not on native HBsAg/ay.
In addition, the ability of the above series of synthetic
polypeptides to bind murine anti-native HBsAg antibodies of d or y
specificity was examined. As shown in Table 1, P72 bound anti-HBs/d
but did not bind to anti-HBs/y; whereas P49 bound anti-HBs/y and
anti-HBs/d to some extent (presumably through a cross-reactive
determinant not related to the common a determinant). The fact that
P49a induced the production anti-polypeptide antibody that reacted
only with the ay subtype and bound anti-HBs/y but not anti-HBs/d
demonstrates the y-specificity of this polypeptide. P73, which is
identical to P72 with the addition of amino-terminal amino acids,
demonstrated primarily d-specificity; however, in immunogenicity
studies a small cross-reactive component was observed. It is
interesting to note that HA inhibition analysis suggested this
component was specific for a determinant common to both subtypes
(i.e., anti-a). The remainder of the synthetic polypeptides used in
this determination induced no anti-peptide antibody cross-reactive
with HBsAg in non-denaturing conditions and did not bind or bound
to a minimal extent anti-native HBsAg antibodies.
These results are in general agreement with those reported by Gerin
et al., Proc. Natl. Acad. Sci. (USA), 80, 2365 (1983) and confirm
the localization of the d and y subtype-specific antibody binding
sites within synthetic polypeptides P72a and P49a, respectively.
These synthetic polypeptides correspond to residues 125-137 of the
amino acid sequence of HBsAg, and although P49a differs from P72a
at four residues, Peterson et al., J. Biol. Chem., 257, 10414
(1982) have suggested that amino acid substitutions at residues 131
and 134 confer subtype-specificity.
The minor and low-titered reactivity of anti-native HBsAg with a
number of the other synthetic polypeptides may result from the
complexity of the antisera that most likely contain specificities
directed to HBsAg degradation products. In support of this
position, antisera to polypeptides P3, P4 and P6 do not react with
native HBsAg, but nonetheless do bind denatured HBsAg [Lerner et
al., Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981)]. The "a-like"
activity of P73 may be explained by the addition of a cysteine
residues to P72, since a cyclic form of a synthetic peptide
corresponding to residues 122 through 137 produced by introduction
of an intrachain disulphide bond has been reported to contain a
conformation-dependent a epitope [Ionescu-Matlo et al., J.
Immunol., 130, 1947 (1983)].
B. Identification of Murine T Cell Epitopes
The polypeptide sequences of HBsAg that are recognized by native
HBsAg primed mouse T cells were identified.
Native HBsAg/adw was purified from the plasma of a single chronic
carrier as described in Peterson et al., J. Biol. Chem., 256, 6975
(1981). P25, a polypeptide subunit of HBsAg, and two tryptic
fragments of P25 designated P25-1 (residues 1-122) and P25-2
(residues 123-226) were prepared from the same HBsAg/adw positive
donor by preparative polyacrylamide gel electrophoresis as also
described in Peterson et al., supra. Synthetic peptides P73, P72,
P49, P6, P5, P5a, P2 and P1 (see FIG. 1) were synthesized according
to the methods described herein. These polypeptides and synthetic
peptides were lyophilized, resuspended in culture media and were
sterilized by gamma radiation (5000 rads).
Culture media used was original Click's Media [as described in
Click et al., Cell Immunol., 3, 264 (1972)] that was modified by
the addition of ten millimolar HEPES
[4(-2-hydroxethyl)-1-piperazinethane-sulfonic acid] and ten
micrograms per milliliter gentamycin and by the substitution of 0.5
percent syngenic normal mouse serum for fetal calf serum. The P25,
P25-1 and P25-2 were not completely soluble in the culture media.
The polypeptides and synthetic peptides suspended in culture media
are referred to herein as antigens and were cultured with harvested
popliteal lymph node (PLN) cells as described below.
C.sub.3 H.Q(H-2.sup.q) is an inbred murine strain that produces
early (10 days) IgG antibodies to both the common a subtype and the
d/y determinants following immunization with HBsAg as described in
Milich et al., J. Immunol., 130, 1395 (1983). Groups of five
C.sub.3 H.Q mice were immunized in the rear footpads with an
emulsion of complete Freund's adjuvant (CFA) and sixteen micrograms
of a pooled purified preparation of HBsAg/ad or HBsAg/ay (obtained
as described earlier). Twelve days later popliteal lymph node (PLN)
cells were harvested and cultured in vitro (2.5.times.10.sup.6
cells per milliliter) with the antigens that were produced as
described above.
The antigens were tested in culture over a dose range, however, the
proliferative responses illustrated in FIG. 3 correspond to the
following in vitro doses: native HBsAg (1.0 micrograms per
milliliter); P25, P25-1, P25-2 (10 micrograms per milliliter); and
synthetic peptides P73, P72, P49, P6, P5, P5a and P2 (100
micrograms per milliliter).
HBsAg specific proliferative response of PLN cells harvested up to
13 days post-immunization was due to proliferating T cells as
described in Milich et al., J. Immunol., 130, 1401 (1983).
Consequently, unfractionated PLN cells were used in the
determinations.
HBsAg specificity was demonstrated by the absence of
antigen-induced proliferation in CFA-primed PLN T cells.
Proliferation was determined by incorporation of tritiated
thymidine (.sup.3 HTdR) into DNA and was expressed as a percent of
the response elicited by the antigen. Assays were repeated on at
least three separate occasions.
The T cell proliferative response is expressed as a percentage of
that induced by the synthetic polypeptide. HBsAg/ad-primed PLN T
cells from C.sub.3 H.Q mice responded in vitro to native HBsAg/adw
almost as well as to the synthetic polypeptides used, and
substantially less to native HBsAg/ay, which represents
proliferation directed towards common group-specific determinant(s)
(FIG. 3a).
Polypeptide P25 induced T cell proliferation to the same extent as
the native HBsAg-adw from which it was prepared. Although
micrograms per milliliter of P25 was required as compared to 1.0
microgram per milliliter of native antigen, the P25 preparation was
not completely soluble in the culture media, and the effective dose
may have been substantially less than 10 micrograms per milliliter.
This is of interest since P25 binds anti-HBs antibody approximately
300-fold less efficiently than the native antigen.
P25-1 induced a better proliferative response than P25-2 (67
percent vs. 45 percent) at 10 micrograms per milliliter, and
significantly greater proliferation at 2.5 micrograms per
milliliter (53 percent vs. 13 percent). The superior proliferative
response induced by P25-1 as compared to P25-2 in this strain was
confirmed by the fact that synthetic polypeptides P73 and P72,
constituents of P25-2, induced minimal proliferative responses;
whereas P6 (residues 95-109), P5 (38-52) and P5a (47-52),
constituents of P25-1, induced significant proliferation in
HBsAg/ad-primed mice (FIG. 3a). Induction of T cell proliferation
by synthetic polypeptides required a 100-fold excess on a weight
basis and a 10.sup.4 -fold excess on a molar basis as compared to
native HBsAg.
It should be emphasized that P6, P5 and P5a do not induce the
production of antibodies cross-reactive with native HBsAg nor do
they bind native anti-HBs, and conversely, P73 and P72 induce only
minimal T cell proliferation in C.sub.3 H.Q mice, yet induce and
bind anti-HBs/d (See Table 1). These results indicate the existence
of distinct T cell and B cell determinants on the same HBsAg
polypeptide. Polypeptide P5a, although only 6 amino acids in
length, induced a greater degree of T cell proliferation than did
polypeptide P5. This may be because P5a is derived from an
extremely hydrophilic region of the amino-terminal fragment (P25-1)
of HBsAg and is considerably more soluble in saline than is
polypeptide P5. As previously discussed, the other large
hydrophilic portion of the polypeptide corresponds to the antibody
binding regions primarily located on the carboxy-terminal fragment
(P25-2).
To determine subtype-specificity of the T cell responses, C.sub.3
H.Q mice were also primed in vivo with HBsAg of the ay subtype. The
proliferative responses to native HBsAg of the ad subtype and to
the adw-derived P25 and tryptic fragments P25-1 and P25-2 were
reduced as compared to HBsAg/ad-primed mice. However, the responses
to synthetic polypeptides P6, P5 and P52 were virtually equivalent
to the responses induced after HBsAg/ad priming (FIG. 3b).
Polypeptides P73 and P72 were not stimulatory for HBsAg/ay-primed T
cells, and P49 did not induce a proliferative response after
priming with either subtype of HBsAg.
These results indicate that P73 and P72 represent subtype-specific
determinants at the T cell and the B cell level. P6, P5 and P5a, on
the other hand, represent common T cell recognition sites present
on both subtypes. This is consistent with the amino acid sequence,
since the P6 and P5a regions are invariable in the HBsAg sequences
determined to date, whereas, the P72 region is variable and amino
acid substitutions in this region dictate subtype-specificity.
Gerin et al., Proc. Natl. Acad. Sci. (USA), 80, 2365 (1983) and
Lerner et al., Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981). The
HBsAg-specificity of these responses was demonstrated by the
absence of proliferation in CFA-primed PLN T cells in response to
HBsAg and its related fragments.
C. Determination of the Role of H-2 Restriction in Mouse T Cell
Recognition Sites
B10.A is an inbred murine strain which only produces an
anti-HBsAg-d/y subtype specific response after primary immunization
as described in Milich et al., J. Exp. Med., 159, 41 (1984) which
is incorporatred herein by reference. Groups of 5 B10.A mice were
primed in vivo with either HBsAg/ad or HBsAg/ay. See FIG. 4. The
immunization protocol, culture conditions and preparation,
concentration of in vitro antigens, and T cell proliferative
testing are the same as described above in section B.
Since the high responder C.sub.3 H.Q strain preferentially
recognizes P25-1, P6 and P5 at the T cell level and produces high
titered anti-HBs/a and anti-HBs/d or y after primary immunization,
it was of interest to examine T cell responses to these antigens in
an intermediate responder strain which only produces
subtype-specific anti-HBs/d or y after primary immunization. The
B10.A strain respresnts such a strain.
B10.A PLN T cells primed with HBsAg/ad responded to native HBsAg/ad
but not at all to native HBsAg/ay (FIG. 3a). Although P25 was
stimulatory for B10.A HBsAg/ad-primed PLN T cells, the responses to
the tryptic fragments P25-1 and P25-2 indicated a preferential
response to P25-2 rather than P25-1 in contrast to the C.sub.3 H.Q
strain. Correspondingly, polypeptides P73 and P72 induced
significant proliferation, whereas polypeptides P6, P5 and P5a were
virtually non-stimulatory for B10.A PLN T cells primed with native
HBsAg/ad or HBs/ay (FIG. 3b).
B10.A mice primed with HBsAg/ay demonstrated only minimal T cell
proliferative response in HBsAg/ay-primed mice (FIG. 3a).
These results demonstrate that B10.A HBsAg-primed T cells
preferentially recognize the subtype-specific regions of the
polypeptide (P25-2 and P72) rather than the group-specific regions
as in the case of C.sub.3 H.Q murine strain. The ability of P72 to
stimulate a d-specific proliferative response and induce and bind
anti-native HBs/d clearly indicates that region 110-137 is
recognized by both T cells and B cells in B10.A mice.
To determine the relevance of the above findings to peptide
immunogenicity and in vivo anti-HBs antibody production, C.sub.3
H.Q and B10.A mice were immunized with P72, an analogue of the d
determinant, and serum anti-peptide and anti-HBs titers were
measured temporally.
As shown in Table 2, following primary immunization with native
HBsAg/ad the C.sub.3 H.Q strain produced subtype and group-specific
anti-HBs. The B10.A strain produced only anti-HBs/d, and to a
lesser degree than the C.sub.3 H.Q strain. However, following
tertiary immunization with P72 the B10.A strain produced a 32-fold
greater anti-P72 response and a 20-fold higher anti HBs/d response
as compared to the C.sub.3 H.Q strain (Table 2).
TABLE 2 ______________________________________ Strain-Dependent In
vivo Antibody Production Following Immunization With Synthetic
Peptide P72 Serum Anti-Polypeptide P72 and Anti-HBs Titers
(RIA).sup.2 Strain Immunogen.sup.1 Anti-P72 Anti-HBs/ --ad
Anti-HBs/ --ay ______________________________________ C.sub.3 H.Q
HBsAg/ --ad (1.degree.) -- 1:2,560 1:320 P72 (1.degree.) 0 0 0 P72
(2.degree.) 1:640 1:8 0 P72 (3.degree.) 1:640 1:8 0 B10.A HBsAg/
--ad (1.degree.) -- 1:320 0 P72 (1.degree.) 1:160 0 0 P72
(2.degree.) 1:2,560 1:20 0 P72 (3.degree.) 1:20,480 1:160 0
______________________________________ .sup.1 Groups of 6 mice were
immunized with 4.0 micrograms of native HBsAg/--ad or 100
micrograms of peptide P72 in CFA intraperitoneally. Peptide
recipient mice were given identical secondary (2.degree.) tertiar
(3.degree.) immunizations at 2week and 4week intervals,
respectively. .sup.2 Pooled serum antibody titers were measured by
solidphase radioimmunoassay (RIA) and expressed as the highest
serum dilution to yield twice the counts of the preimmunization
sera.
It should be noted that C.sub.3 H.Q mice immunized with P72
conjugated to a carrier protein produced vigorous anti-P72 and
anti-HBs/d responses. Reduced immunogenicity of P72 in the C.sub.3
H.Q strain is consistent with the inability of P72 to induce a T
cell proliferative response in HBsAg/ad-primed C.sub.3 H.Q mice.
These results illustrate that the high responder status of the
C.sub.3 H.Q strain is not mediated through T cell recognition of
the subtype-specific d determinant as represented by P72. In
contrast, the B10.A strain, which demonstrates P72-induced T cell
proliferation following HBsAg/ad immunization, was capable of
responding to P72 immunization with the production of significant
concentrations of anti-P72 and anti-HBs/d antibodies. Therefore,
the immunogenicity of synthetic peptide analogues of HBsAg is
dependent on the requirement for both T cell and B cell
determinants; and the recognition of the T cell determinant is
dictated by the H-2 genotype of the responding murine strain.
D. Determination of Synthetic Peptides That Elicit HBsAg-Specific T
Cell Proliferation in Mice and are Recognized by Human Vaccine
Recipient HBsAg Primed T Cells
Peripheral blood lymphocytes from two human HBsAg/ad vaccine
recipients (Haptavax, Merck & Co., Rahway, N.J.) designated
"DM" and "PW" and an unimmunized volunteer designated "CL" were
compared for T cell responsiveness to native HBsAg/ad, native
HBsAg/ay, and a series of synthetic peptide analogues of HBsAg.
As shown in Table 3, peripheral blood lymphocytes from one HBsAg
vaccine recipient (DM) responded to native HBsAg of both subtypes
and to polypeptides P72 and P5. However, the responses elicited by
native HBsAg/ad and by polypeptide P72 were significantly greater
than those elicited by native HBsAg/ay and by polypeptide P5 in
terms of stimulation index and dose response.
In contrast, PBL from (PW) responded equally well to both native
HBsAg subtypes, and correspondingly polypeptides P1 (residues
48-81) and P5 induced proliferative responses, whereas polypeptide
72 did not. The other synthetic peptides tested were not
stimulatory nor did any of the antigens stimulate PBL obtained from
the nonimmunized control (CL).
Similar to the findings of the murine model, at least two patterns
of T cell specificity were observed in human responses. One pattern
is characteristic of T cell recognition of distinct determinants
(P1 and P5), which do not induce the production of or bind to
anti-HBs antibodies. The other pattern involves recognition by T
cells of a subtype-specific region that may overlap with B cell
determinants.
TABLE 3
__________________________________________________________________________
PBL.sup.1 Proliferative Responses of Human HBsAg Vaccine Recipients
Challenged In Vitro With Synthetic Peptide Analogues And Native
HBsAg Anti-HBs status PBL Proliferative Responses (.sup.3 H-TdR;
CPM and SI).sup.2 HBsAg/adw (HA Titer) In Vitro Antigens.sup.3
Vaccine HBsAg/ad HBsAg/ay Media HBsAg/ad HBsAg/ay P72 P49 P6 P5 P3
P2 P1
__________________________________________________________________________
(DM) + 1:1600 1:800 3100 31,794 20,656 45,776 3376 5583 11,648 4442
5320 3500 (10.3) (6.7) (14.8) (1.1) (1.8) (3.8) (1.4) (1.7) (1.1)
(PW) + 1:8,192 1:1024 5000 14,665 16,958 6325 6330 4784 10,500 5683
ND 22,102 (3.0) (3.4) (1.3) (1.3) (0.9) (2.1) (1.1) (4.4) (CL) - 0
0 2475 2426 3638 3069 2723 2277 3316 ND.sup.4 ND 2000 (1.0) (1.5)
(1.2) (1.1) (0.9) (1.3) (0.8)
__________________________________________________________________________
.sup.1 PBL = Peripheral blood lymphocytes. .sup.2 Human
proliferative responses were measured using peripheral blood
lymphoyctes (PBL) and culture conditions modified from the murine
assay a described in LerouxRoels et al., (in press). The .sup.3
HTdR incorporatio is expressed as counts per minute (CPM) and
stimulation index (SI). A stimulation index is the ratio of the
stimulation induced by the test antigen (measured as counts per
minute) to the stimulation induced by the control media (also
measured as counts per minute). .sup.3 Antigens were used over a
wide dose range; proliferative responses to 1.0 microgram per
milliliter of native HBsAg and 100.0 micrograms per milliliter of
peptide analogues are shown. Underscored responses were greater
than two times the media control through at least fourfold
dilutions. .sup.4 ND = Not Determined.
V. Materials and Methods
A. Materials
The C.sub.3 HQ and B10.A inbred murine strains and New Zealand
white rabbits were obtained from the Research Institute of Scripps
Clinic, La Jolla, Calif. The B10.T(6R) strain was provided by Dr.
Hugh McDevitt (Stanford University, Palo Alto, Calif.). Female mice
between 6 and 12 weeks of age at the initiation of the studies were
used in all studies.
Pooled preparations of HBsAg/ad and HBsAg/ay were provided by Dr.
Robert Louie (Cutter Laboratories, Berkeley, Calif.). These
preparations were purified by Cutter Laboratories from human plasma
by a combination of standard procedures including
ultracentrifugation, ammonium sulfate precipitation, pepsin
digestion and gel chromatography. The HBsAg preparations were free
of contaminating human serum proteins as tested by Ouchterlony
analysis and immunoelectrophoresis versus goat anti-human serum
[Milich et al., J. Immunol., 129, 320 (1982) which is incorporated
herein by reference].
Native HBsAg/adw was purified from the plasma of a single chronic
carrier donor by methods previously described by Peterson et al.,
J. Biol. Chem., 256, 6975 (1981). The structural polypeptide (P-25)
and the tryptic fragments P-25-1 (residues 1-122) and P-25-2
(residues 123-226) were prepared from this same HBsAg/adw positive
donor by preparative polyacrylamide gel electrophoresis also as
described by Peterson et al., supra. The synthetic peptides shown
in FIG. 1 were synthesized by the solid-phase methods described
herein. The polypeptides and tryptic fragments were lyophilized,
resuspended in culture media as previously described and were
sterilized by gamma irradiation (5000 rads).
B. Immunization
Anti-polypeptide antibodies were produced in rabbits. Polypeptides
were coupled to keyhole limpet hemocyanin (KLH) through the
existent or added cysteine of the polypeptide by using
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling
reagent [See Section VI(c)]. Rabbits were immunized with
polypeptide-KLH conjugates according to the following schedule: (1)
200 micrograms of polypeptide in complete Freund's adjuvant (CFA)
administered subcutaneously on day 0; (2) 200 micrograms of
polypeptide in incomplete Freund's adjuvant (IFA) on day 14; and
(3) 200 micrograms of polypeptide with 4 milligrams alum
administered intraperitoneally on days 21 and 91. The animals were
bled 15 weeks after the first injection. Polypeptides 1, 72 and 73
were injected without KLH. The above weights of the polypeptides do
not include the weights of the carriers.
To study in vivo antibody production in mice, groups of mice were
immunized with 4.0 micrograms native HBsAg/ad or 100 micrograms p72
in CFA by intraperitoneal injection. Peptide recipient mice were
given identical secondary and tertiary immunizations at 2 week and
4 week intervals. In vivo priming for the lymph node proliferative
assay was accomplished by injection of a total of 16.0 micrograms
HBsAg in CFA in a volume of 80 microliters into the two hind
footpads of the recipient mice.
C. Measurement of anti-HBs.
Anti-HBs antibodies induced by immunization with a synthetic
polypeptide or native HBsAg and anti-polypeptide antibodies induced
by polypeptide immunization were measured by two methods. Murine
sera were evaluated for anti-HBs and anti-polypeptide reactivity in
an indirect, immunoglobulin class-specific, radiommunoasay (RIA)
utilizing solid-phase HBsAg (ad or ay subtype) or synthetic
peptides, goat anti-mouse IgG, and were developed with .sup.125
I-labeled, swine anti-goat Ig as described in Milich et al., J.
Immunol., 129, 320 (1982).
To analyze rabbit sera for anti-HBs activity, a hemagglutination
(HA) system was used. Human type `O`, Rh negative red blood cells
were coated with HBsAg (ad or ay subtype) by the chromic chloride
method as described in Vyas et al., Science, 170, 332 (1970) which
is incorporated herein by reference. The coated cells were added to
0.25 milliliters of serially diluted test sera in microtiter
`V`-bottom plates. All anti-HBs assays were performed in 5-10
percent normal human sera to neutralize any possible antibodies to
contaminating human plasma proteins that may not have been removed
from the HBsAg preparation by the purification procedures
utilized.
D. Lymph node proliferation assay.
Groups of 5 mice were immunized in the hind footpads with an
emulsion of CFA and 16 micrograms HBsAg (ad or ay subtype). Twelve
days later popliteal lymph node (PLN) cells were harvested and
cultured in vitro to a concentration of 5.times.10.sup.5 cells with
various challenge antigens. The in vitro antigens included native
HBsAg [ad (pooled), ay (pooled) or adw from a single donor];
polypeptide P25; tryptic fragments P-25-1 and P-25-2; and the
synthetic polypeptide of the present invention (P73, P72, P49, P6,
P5, P5a, P2, P1).
Draining popliteal lymph node cells were aseptically removed from
each mouse and teased to yield a single cell suspension. The cells
were washed twice with a balanced salt solution (BSS) containing
phosphate-buffered saline (pH 7.2). The cells were resuspended in
Click's medium containing BSS, L-glutamine, sodium pyruvate,
antibiotics, 2-mercaptoethanol, essential and non-essential amino
acids and vitamins. [See Click et al., Cell Immunol., 3, 264
(1972).] Click's medium, was however modified by the addition of 10
millimolar HEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic
acid) and gentamycin (10 micrograms per milliliter) and by the
substitution of 0.5 percent syngeneic normal mouse serum for fetal
calf serum.
The antigens were tested in culture over a dose range. However, the
proliferative responses shown in FIGS. 3 and 4 correspond to the
following in vitro doses: native 100 micrograms per milliliter.
Viable lymph node cells (4.times.10.sup.5) in 0.1 milliliter of
medium were placed in flat-bottom microtiter wells (Falcon 3072,
Falcon Plastics, Inc.) with: (a) 0.1 ml. of HBsAg of the ad or ay
subtype (2.0 to 0.6 micrograms per milliliter), (b) culture medium
and ovalbimin (200 micrograms per milliliter as a negative control,
or (c) purified protein derivative (PPD-50 micrograms per
milliliter) as a positive control.
Cultures were incubated for 5 days at 37 degrees C. in a humidified
atmosphere containing 5 percent carbon dioxide in air.
On the fourth day, each culture was pulsed with microcurie .sup.3
H-thymidine (.sup.3 HTdR) (6.7 Ci/millimole, New England Nuclear,
Boston, Mass.) 16 to 18 hours before harvesting. Proliferation was
determined by the incorporation of .sup.3 HTdR into DNA. Specific
proliferation as a stimulation index (SI) that equals the counts
per minute (cpm) of the test antigen divided by the cpm of the
media control. It was demonstrated previously that the
HBsAg-specific proliferation response of draining PLN cells
harvested up to 13 days post-immunization is due to proliferating T
cells [Milich et al., J. Immunol, 130, 1401 (1983)]. Therefore,
unfractionated PLN cells were used in experiments reported
herein.
VI. Peptide Syntheses and Selection
A. Synthesis of Polypeptides
The polypeptides of this invention were chemically synthesized by
solid-phase methods as described in Merrifield et al., J. Am. Chem.
Soc., 85, 2149 (1963) and Houghten et al., Int. J. Peptide Protein
Research, 16, 311 (1980). The relatively short polypeptides used
herein substantially correspond to antigenic determinants of
HBsAg.
FIG. 1 shows the 226 amino acid residue sequence of HBsAg. The
amino acid residue sequences of the preferred synthetic
polypeptides described herein are shown in FIGS. 1 and 2. In
certain instances, a cysteine residue was added to the
amino-terminus or to the carboxy-terminus of some of the
polypeptides to assist in coupling to a protein carrier as
described below. The compositions of all polypeptides were
confirmed by amino acid analysis.
Generally, an immunogen or synthetic polypeptide is made by the
steps of providing a plurality of amino acids that correspond to
the amino acid residues of an antigenic determinant domain of HBsAg
and synthesizing those amino acids into a polypeptide that has a
peptide sequence corresponding to the peptide sequence of that
antigenic determinant. The produced synthetic polypeptide can be
used to produce a vaccine, usually by linking it to a carrier to
form a conjugate and then dispersing an effective amount of the
conjugate in a physiologically tolerable diluent.
The polypeptides are preferably synthesized according to the
above-referenced solid phase methods using a cysteine resin. See
Merrifield et al., supra. The side chains on individual amino acids
are protected as follows: Arg-tosyl, Ser-, Thr-, Glu-and
Asp-O-benzyl; Tyr-O-bromobenzyloxy carbamyl; Trp-N-formyl. The
N-formyl group on the Trp residues is removed after cleavage of the
peptide from the resin support by treatment with 1.0 molar ammonium
bicarbonate at a peptide concentration of 1.0 milligram/milliliter
for 16 hours at the room temperature. Yamashiro et al., J. Org.
Chem., 38, 2594-2597 (1973). The efficiency of coupling at each
step can be monitored with ninhydrin or picric acid and is
preferably greater than 99 percent in all cases. See Gisin, Anal.
Chem. Acta, 58, 248-249 (1972) and Kaiser, Anal. Biochem., 34,
595-598 (1980).
Throughout the application, the phrase "immunologically corresponds
substantially" in its various grammatical forms is used herein and
in the claims in relation to polypeptide sequences to mean that the
polypeptide sequence described induces production of antibodies
that bind to the polypeptide and (a) bind to the antigenic
determinant of native HBsAg for polypeptides 49, 49a, 72 and 72a or
(b) induce T cell proliferation for polypeptides 1, 5, 5a and 6.
Thus, the peptides of this invention function immunologically as do
the corresponding portions of the HBsAg molecules while also being
capable of inducing the production of antibodies to themselves.
The term "substantially corresponds" in its various grammatical
forms is used herein and in the claims in relation to polypeptide
sequences to mean the polypeptide sequence described plus or minus
up to three amino acid residues at either or both of the amino- and
carboxy-termini and containing only conservative substitutions in
particular amino acid residues along the polypeptide sequence.
The term "conservative substitution" as used above is meant to
denote that one amino acid residue has been replaced by another,
biologically similar residue. Examples of conservative
substitutions include the substitution of one hydrophobic residue
such as Ile, Val, Leu or Met for another, or the substitution of
one polar residue for another such as between Arg and Lys, between
Glu and Asp or between Gln and Asn, and the like.
In some instances, the replacement of an ionic residue by an
oppositely charged ionic residue such as Asp by Lys has been termed
conservative in the art in that those ionic groups are thought to
merely provide solubility assistance. In general, however, since
the replacements discussed herein are on relatively short synthetic
polypeptide antigens, as compared to a whole protein, replacement
of an ionic residue by another ionic residue of opposite charge is
considered herein to be "radical replacement", as are replacements
between nonionic and ionic residues, and bulky residues such as
Phe, Tyr or Trp and less bulky residues such as Gly, Ile and
Val.
The terms "nonionic" and "ionic" residues are used herein in their
usual sense to designate those amino acid residues that normally
either bear no charge or normally bear a charge, respectively, at
physiological pH values. Exemplary nonionic residues include Thr
and Gln, while exemplary ionic residues include Arg and Asp.
The word "antigen" has been used historically to designate an
entity that is bound by an antibody and to designate the entity
that induces the production of the antibody. More current usage
limits the meaning of antigen to that entity bound by an antibody,
while the word "immunogen" is used for the entity that induces
antibody production. In some instances, the antigen and immunogen
are the same entity as where a synthetic polypeptide is utilized to
induce production of antibodies that bind to the polypeptide.
However, the same polypeptide (P49a) can also be utilized to induce
antibodies that bind to a whole protein such as HBsAg, in which
case the polypeptide is both immunogen and antigen, while the HBsAg
is an antigen. Where an entity discussed herein is both immunogenic
and antigenic, it will generally be termed an antigen.
B. Preparation of Polymers
The polypeptides of the present invention can be connected together
to form an antigenic polymer (synthetic multimer) comprising a
plurality of the polypeptide repeating units. Such a polymer has
the advantages of increased immunological reaction and where
different polypeptides are used to make up the polymer, the
additional ability to induce antibodies that immunoreact with
several antigenic determinants of HBsAg.
A polymer (synthetic multimer) can be prepared by synthesizing the
polypeptides as discussed above and by adding cysteine residues at
both the amino- and carboxy-termini to form a "diCys-terminated"
polypeptide. Thereafter, in a typical laboratory preparation, 10
milligrams of the diCys polypeptide (containing cysteine residues
in un-oxidized form) are dissolved in 250 milliliters of 0.1 molar
ammonium bicarbonate buffer. The dissolved diCys-terminated
polypeptide is then air oxidized by stirring the resulting solution
gently for a period of about 18 hours, or until there is no
detectable free mercaptan by the Ellman test. [See Ellman, Arch.
Biochem. Biophys., 82, 70 (1959).]
The polymer (synthetic multimer) so prepared contains a plurality
of the polypeptides of this invention as repeating units. Those
polypeptide repeating units are bonded together by oxidized
cysteine residues.
C. Coupling of Polypeptides to Protein Carriers
The synthetic polypeptides were coupled to keyhole limpet
hemocyanin (KLH) or tetanus toxoid (TT) by either of the following
two methods. In the first method, the carrier was activated with
m-maleimidobenzoyl-N-hydroxysuccinimide ester and was subsequently
coupled to the polypeptide through a cysteine residue added to the
amino- or carboxy-terminus of the polypeptide, as described in Liu
et al., Biochem., 80, 690 (1979). In the second method, the
polypeptide was coupled to the carrier through free amino groups,
using a 0.04 percent glutaraldehyde solution as is well known. See,
for example, Klipstein et al., J. Inpect. Disc., 147, 318
(1983).
As discussed before, cysteine residues added at the amino- and/or
carboxy-terminii of the synthetic polypeptide have been found to be
particularly useful for forming conjugates via disulfide bonds and
Michael addition reaction products, but other methods well known in
the art for preparing conjugates can also be used. Exemplary
additional binding procedures include the use of dialdehydes such
as glutaraldehyde (discussed above) and the like, or the use of
carbodiimide technology as in the use of a water-soluble
carbodiimide, e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,
to form amide links to the carrier.
Useful carriers are well known in the art and are generally
proteins themselves. Exemplary of such carriers are keyhole limpet
hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine
serum albumin or human serum albumin (BSA or HSA, respectively),
red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid,
cholera toxoid as well as polyamino acids such as
poly(D-lysine:D-glutamic acid), and the like.
As is also well known in the art, it is often beneficial to bind
the synthetic polypeptide to its carrier by means of an
intermediate, linking group. As noted above, glutaraldehyde is one
such linking group. However, when cysteine is used, the
intermediate linking group is preferably an m-maleimidobenzoyl
N-hydroxysuccinimide ester (MBS). MBS is typically first added to
the carrier by an ester-amide interchange reaction. Thereafter, the
above Michael reaction can be followed, or the addition can be
followed by addition of a blocked mercapto group such as
thiolacetic acid (CH.sub.3 COSH) across the maleimido-double bond.
After cleavage of the acyl blocking group, and a disulfide bond is
formed between the deblocked linking group mercaptan and the
mercaptan of the added cysteine residue of the synthetic
polypeptide.
The choice of carrier is more dependent upon the ultimate intended
use of the antigen than upon the determinant portion of the
antigen, and is based upon criteria not particularly involved in
the present invention. For example, if a vaccine is to be used in
animals, a carrier that does not generate an untoward reaction in
the particular animal should be selected. If a vaccine is to be
used in man, then the overriding concerns involve the lack of
immunochemical or other side reaction of the carrier and/or the
resulting antigen, safety and efficacy--the same considerations
that apply to any vaccine intended for human use.
VII. Immunization Procedures
The inocula or vaccines used herein contain an effective amount of
polypeptide alone, as a polymer of individual polypeptides linked
together through oxidized cysteine residues or as a conjugate
linked to a carrier. The effective amount of polypeptide per
inoculation depends, among other things, on the species of animal
inoculated, the body weight of the animal and the chosen
inoculation regimen as is well known. Vaccines are typically
prepared from the dried solid polypeptide or polypeptide polymer by
suspending the polypeptide or polypeptide polymer in water, saline
or adjuvant, or by binding the polypeptide to a carrier and
suspending the carrier-bound polypeptide (conjugate) in a similar
physiologically tolerable diluent such as an adjuvant (as
previously described).
These inocula typically contain polypeptide concentrations of about
20 micrograms to about 500 milligrams per inoculation. The stated
amounts of polypeptide refer to the weight of polypeptide without
the weight of a carrier, when a carrier was used.
The vaccines also contained a physiologically tolerable
(acceptable) diluent such as water, phosphate-buffered saline or
saline, and further typically include an adjuvant. Adjuvants such
as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant
(IFA) and alum are materials well known in the art, and are
available commercially from several sources.
Vaccine stock solutions were prepared with CFA, IFA or alum as
follows: An amount of the synthetic polypeptide, polymeric
polypeptide or conjugate sufficient to provide the desired amount
of polypeptide per inoculation was dissolved in phosphate-buffered
saline (PBS) at a pH value of 7.2. Equal volumes of CFA, IFA or
alum were then mixed with the polypeptide solution to provide a
vaccine containing polypeptide, water and adjuvant in which the
water-to-oil ratio was about 1:1. The mixture was thereafter
homogenized to provide the vaccine stock solution.
Rabbits were injected subcutaneously and intraperitoneally, as
previously described, with a vaccine comprising 200 to 400
micrograms of a polypeptide conjugate emulsified in complete
Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) or alum
(5 milligrams per milliliter in each instance) on days 0, 14 and
21, respectively. Each inoculation (immunization) consisted of four
injections of the inoculum. Mice were immunized in a similar way
using one tenth of the above dose per injection.
Animals were bled 7 and 14 days after the last injection. In some
cases, the animals received booster injections in alum, and were
bled thereafter as necessary. Control pre-immune serum was obtained
from each animal by bleeding just before the initial
immunization.
Inoculum stock solutions can also be prepared with keyhole limpet
hemocyanin (KLH), KLH in IFA (incomplete Freund's adjuvant),
KLH-alum absorbed, KLH-alum absorbed-pertussis, edestin,
thyroglobulin, tetanus toxoid, tetanus toxoid in IFA, cholera
toxoid and cholera toxoid in IFA.
Upon injection or other introduction of the antigen or vaccine into
the host, the immune system of the host responds by producing large
amounts of antibody to the antigen. Since the specific antigenic
determinant of the manufactured antigen, i.e., the antigen formed
from the synthetic polypeptide and the carrier immunologically
corresponds substantially to the determinant of the natural antigen
of interest, the host becomes immune to the natural antigen. In the
case where the invention is used as a vaccine, this is the desired
result.
VIII. Delayed-Type Hypersensitivity
(Skin Reaction Test)
The previously described diagnostic systems and assays are based on
in vitro assays. Although particular steps of the assays can be
carried out in vivo, the actual immune response is measured in
tissue culture. The present invention, however, can also be applied
to diagnostic systems involving the in vivo measurement of T cell
responses. One example of such a system is a delayed-type
hypersensitivity (DTH) reaction or what is more commonly known as a
skin reaction test.
A DTH reaction can only occur in an individual previously exposed
(sensitized) to a given antigen. The first exposure of an
individual to the antigen produces no visible change, but the
immune status of the individual is altered in that hypersensitivity
to renewed exposure to that antigen results. Thus, upon intradermal
or subcutaneous injection of the antigen (preferably in a buffered
saline solution) a characteristic skin lesion develops at the
injection site--a lesion that would not develop after a first
antigen exposure. Because the response to the second (or challenge)
antigen inoculum is typically delayed by 24 to 48 hours, the
reaction is referred to as delayed-type hypersensitivity.
In humans, exposure to a sensitizing antigen takes place upon
contact with the microorganism responsible for the disease (e.g.,
tuberculin from Mycobacterium tuberculosis, typhoidin from
Salmonella typhi and abortin from Brucella abortus), and
sensitization occurs as a result of a chronic infection. In
animals, sensitization can be achieved by inoculation of an antigen
emulsified in water, saline or an adjuvant.
In both humans and animals, hypersensitivity is tested in vivo by
the injection of the antigen dissolved in a physiologically
tolerable diluent such as saline solution into the skin (either
intradermally or subcutaneously). DTH is usually a more sensitive
diagnostic assay than the determination or measurement of the
amount of antibody produced to an antigen. For example, only minute
amounts of protein (a few hundred micrograms) are necessary for DTH
sensitization of a mouse, while a much larger dose is needed to
induce antibody production.
Since the polypeptides of the present invention stimulate the
proliferation of human and murine T cells following immunization
(sensitization) with active HBsAg or with polypeptides 49, 49a, 72
and 72a, a skin reaction test was developed using one or more of
the present synthetic polypeptides as a challenge antigen.
Selected murine strains are immunized with native HBsAg emulsified
on an adjuvant such as complete Freund's adjuvant (CFA) by
intradermal injection in the flank. In experimental situations, DTH
sensitization usually occurs only when the sensitizing antigen is
administered in adjuvant, preferably the complete type that
includes bacilli of tuberculosis.
Seven days after immunization, the mice are challenged by
intradermal inoculation in the ear or in the footpad with a
predetermined amount of an antigen including (a) native HBsAg or
(b) one or more of the present synthetic polypeptides in a known
volume of phosphate-buffered saline (PBS). Control mice are
inoculated intradermally with the same volume of PBS not including
the antigen. Additional controls include mice immunized with only
CFA.
Thickening of the tissue at the antigen-injection site relative to
the control sites is evidence of a DTH reaction. Thus, the
thickness of the ears and footpads is measured before challenge
with the antigen and at 4, 24 and 48 hours after challenge.
Results demonstrate that the synthetic polypeptides of the present
invention may be useful in an in vivo murine diagnostic system for
the presence of a cell mediated immune response to HBsAg.
After the safety and effectiveness of the above polypeptides are
shown in animal studies, the polypeptides can be used as challenge
antigens in human skin reaction tests for recipients of HBsAg
vaccines. The polypeptides are synthesized as previously described,
purified by high pressure liquid chromatography (HPLC) techniques,
sterilized and pyrogen-tested.
Since the T cell proliferative responses of human HBsAg vaccine
recipients can be quite variable relative to polypeptide
specificity, vaccine recipients and individuals serving as
unvaccinated controls are challenged with a series of polypeptides.
The kinetics and optimal antigen dose can be determined in the
vaccine recipient group using the results from the animal studies
as a guideline.
HBV acute and chronically infected individuals can also be studied
for HBsAg-specific T cell sensitization using synthetic
polypeptides as antigens for a skin reaction test.
In each instance, the challenge antigen is administered by
intradermal injection of the particular polypeptide in a
physiologically acceptable solution (about 1 milliliter) into the
volar surface of the forearm. Use of a 25- or 27-gauge needle
usually assures intradermal rather than subcutaneous administration
of the antigen. Subcutaneous injection can lead to dilution of the
antigen in tissues and can produce a false-negative test. The
injection sites are then observed for erythema (skin reddening) and
induration (swelling) at 4, 24 and 48 hours post-challenge.
The foregoing is intended as illustrative of the present invention
but is not limiting. Numerous variations and modifications can be
made without departing from the spirit and scope of the novel
concepts of the invention. It should be understood that no
limitation with respect to the specific compositions and uses
described herein is intended or should be inferred.
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